Detection of IgG3 antibodies specific to the human immunodeficiency virus type 1 (HIV-1) p24 protein as marker for recently acquired infection.

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

Incidence and risk factors for Dengue virus (DENV) infection in the first 2 years of life in a Brazilian prospective birth cohort.

This study assessed the incidence and risk factors for dengue virus (DENV) infection among children in a prospective birth cohort conducted in the city of Recife, a hyperendemic dengue area in Northeast Brazil. Healthy pregnant women (n = 415) residing in Recife who agreed to have their children followed were enrolled. Children were followed during their first 24 months of age (May/2011-June/2014), before the 2015 Zika virus outbreak. DENV infection was detected by reverse-transcriptase polymerase chain reaction and/or serology (anti-DENV IgM/IgG). The incidence rates per 1000 person-years (py) and its association with risk factors by age bands (0-12, >12-30 months) were estimated through Poisson regression models. Forty-nine dengue infections were detected; none progressed to severe forms. The incidence rates were 107·6/1000py (95% CI 76·8-150·6) and 93·3/1000py (95% CI 56·1-154·4) in the first and second years of age, respectively. Male children (risk ratios (RR) = 2·33; 95% CI 1·09-4·98) and those born to DENV-naïve mothers (RR = 2·42; 95% CI 1·01-5·80) were at greater risk of infection in the first year of age. In the second year, children born to Caucasian/Asian descent skin colour mothers had a threefold higher risk of infection (RR = 3·34; 95% CI: 1·08-10·33). These data show the high exposure of children to DENV infection in our setting and highlight the role of biological factors in this population's susceptibility to infection.

Development of potent class II transactivator gene delivery systems capable of inducing de novo MHC II expression in human cells, in vitro and ex vivo.

Class II transactivator (CIITA) induces transcription of major histocompatibility complex (MHC) II genes and can potentially be used to improve genetic immunotherapies by converting non-immune cells into cells capable of presenting antigens to CD4+ T cells. However, CIITA expression is tightly controlled and it remains unclear whether distinct non-immune cells differ in this transactivator regulation. Here we describe the development of gene delivery systems capable of promoting the efficient CIITA expression in non-immune cell lines and in primary human cells of an ex vivo skin explant model. Different human cell types undergoing CIITA overexpression presented high-level de novo expression of MHC II, validating the delivery systems as suitable tools for the CIITA evaluation as a molecular adjuvant for gene therapies.

Draft Genome Sequence of the Probiotic Yeast Saccharomyces cerevisiae var. boulardii Strain ATCC MYA-796.

Saccharomyces boulardii is the only yeast approved as a probiotic for human consumption. Here, we report the draft genome sequence of the strain ATCC MYA-796, derived from the French Ultra Levure probiotic drug. The genome has a size of 11.6 Mb with 5,305 putative open reading frames predicted.

Force of infection of dengue serotypes in a population-based study in the northeast of Brazil.

This study investigated anti-dengue serotype-specific neutralizing antibodies in a random sample of dengue IgG-positive individuals identified in a survey performed in a hyperendemic setting in northeastern Brazil in 2005. Of 323 individuals, 174 (53.8%) had antibodies to dengue virus serotype 1 (DENV-1), 104 (32.2%) to DENV-2 and 301 (93.2%) to DENV-3. Monotypic infections by DENV-3 were the most frequent infection (35.6%). Of 109 individuals aged <15 years, 61.5% presented multitypic infections. The force of infection estimated by a catalytic model was 0.9%, 0.4% and 2.5% person-years for DENV-1, DENV-2 and DENV-3, respectively. By the age of 5 years, about 70%, 30% and 40% of participants were immune to DENV-3, DENV-2 and DENV-1, respectively. The data suggest that infection with DENV-1, -2 and -3 is intense at early ages, demonstrating the need for research efforts to investigate dengue infection in representative population samples of Brazilian children during early infancy.

Immune transcript variations among Aedes aegypti populations with distinct susceptibility to dengue virus serotype 2.

The innate immune response of insects is one of the factors that may dictate their susceptibility to viral infection. Two immune signaling pathways, Toll and JAK-STAT, and the RNA interference (RNAi) pathway are involved in Aedes aegypti responses against dengue virus (DENV), however natural differences in these antiviral defenses among mosquito populations have not been studied. Here, two field Ae. aegypti populations from distinct ecological environments, one from Recife and the other from Petrolina (Brazil), and a laboratory strain were studied for their ability to replicate a primary isolate of dengue virus serotype 2 (DENV-2). Virus infectivity and replication were determined in insect tissues collected after viral exposure through reverse-transcription real time PCR (RT-PCR). The expression of a transcript representing these defense mechanisms (Toll, JAK-STAT and RNAi) in the midgut and fat body was studied with RT-PCR to evaluate variations in innate immune mechanisms possibly employed against DENV. Analyses of infection rates indicated that the field populations were more susceptible to DENV-2 infection than the lab strain. There were distinct expression patterns among mosquito populations, in both control and infected insects. Moreover, lower expression of immune molecules in DENV-2-infected insects compared to controls was observed in the two field populations. These results suggest that natural variations in vector competence against DENV may be partly due to differences in mosquito defense mechanisms, and that the down-regulation of immune transcripts after viral infection depends on the insect strain.

Immune responses to T-cell epitopes of SARS CoV-N protein are enhanced by N immunization with a chimera of lysosome-associated membrane protein.

In our previous study by Gupta et al, dominant T-cell epitopes of SARS CoV-N(N) protein were predicted by software. The spectrum of interferon (IFN)-gamma responses of Balb/c mice immunized against two different forms of SARS CoV-N plasmid was then analyzed. A cluster of dominant T-cell epitopes of SARS CoV-N protein was found in the N-terminus (amino acids 76-114). On the basis of this study, four different plasmids were constructed: (i) DNA encoding the unmodified N (p-N) or N(70-122) (p-N(70-122)) as an endogenous cytoplasmic protein or (ii) DNA encoding a lysosome-associated membrane protein (LAMP) chimera with N (p-LAMP/N) or N(70-122) (p-LAMP/N(70-122)). The immune responses of mice to these four constructs were evaluated. The results showed marked differences in the responses of the immunized mice. A single priming immunization with the p-LAMP/N construct was sufficient to elicit an antibody response. Enzyme-linked immunospot (ELISpot) assay indicated that p-LAMP/N(70-122) and p-LAMP/N plasmids both elicited a greater IFN-gamma response than p-N. p-N and p-N(70-122) constructs induced low or undetectable levels of cytokine secretion. We also found that the p-LAMP/N(70-122) construct promoted a long-lasting T-cell memory response without an additional boost 6 months after three immunizations. These findings show that DNA vaccines, even epitope-based DNA vaccines using LAMP as chimera, can elicit both humoral and cellular immune responses.

In silico identification of new genetic variations as potential risk factors for Alzheimer's disease in a microarray-oriented simulation.

Genomic and proteomic studies of neurodegenerative disorders require complementary approaches to integrate the massive amount of data generated in high throughput experimental procedures. We propose a Bioinformatics pipeline in which expression studies guide the selection of candidate genes that should be screened for potential new genetic variations from a public expressed site tags (ESTs) database. Motivated by the former interest of our group in genetic polymorphisms involved with the immune system, we selected five genes from a previous expression microarrays study of hippocampal cornu ammonis (CA1) area of Alzheimer's Disease subjects (AD). The CLCbio Workbench Combined version 3.6.2. was initially used to build ESTs and mRNA files retrieved respectively from the Goldenpath of University of California Santa Cruz (UCSC) and National Center for Biotechnology Information (NCBI) databases and latter to perform multiple batches of Smith-Waterman alignments. A total of 116 ESTs sequences were selected after proper stringent parameters were applied to the first set of mismatches. The annotation revealed various classes of variations, most of them deletions (176). Amongst this specific group, some were frameshift deletions (35) and the virtual translation of a few others (5) were predicted to induce no change other than a single aminoacid removal, with no subsequent repercussions at the protein sequence. In addition, the analysis identified transitions (three), transversions (52), synonymous (41), non-synonymous (12), and deletions in 36 ESTs located in Untranslated Regions -UTRs (Supplementary data). Deletions are often associated to major genetics syndromes with dysmorphic features. However, various recent studies show that common microdeletions might be highly associated with common neuropsychiatric disorders such as schizophrenia, autism, mental retardation, or even in various ethnicities, detected in whole genome sequencing experiments. A virtual validation confirmed that some of the variations identified were previously reported and confirmed in DNA samples, showing that this method is a feasible way to detect genetic variations that merit further exploration in AD genetic risk factor association studies.

Conservation and variability of dengue virus proteins: implications for vaccine design.

BACKGROUND: Genetic variation and rapid evolution are hallmarks of RNA viruses, the result of high mutation rates in RNA replication and selection of mutants that enhance viral adaptation, including the escape from host immune responses. Variability is uneven across the genome because mutations resulting in a deleterious effect on viral fitness are restricted. RNA viruses are thus marked by protein sites permissive to multiple mutations and sites critical to viral structure-function that are evolutionarily robust and highly conserved. Identification and characterization of the historical dynamics of the conserved sites have relevance to multiple applications, including potential targets for diagnosis, and prophylactic and therapeutic purposes. METHODOLOGY/PRINCIPAL FINDINGS: We describe a large-scale identification and analysis of evolutionarily highly conserved amino acid sequences of the entire dengue virus (DENV) proteome, with a focus on sequences of 9 amino acids or more, and thus immune-relevant as potential T-cell determinants. DENV protein sequence data were collected from the NCBI Entrez protein database in 2005 (9,512 sequences) and again in 2007 (12,404 sequences). Forty-four (44) sequences (pan-DENV sequences), mainly those of nonstructural proteins and representing approximately 15% of the DENV polyprotein length, were identical in 80% or more of all recorded DENV sequences. Of these 44 sequences, 34 ( approximately 77%) were present in >or=95% of sequences of each DENV type, and 27 ( approximately 61%) were conserved in other Flaviviruses. The frequencies of variants of the pan-DENV sequences were low (0 to approximately 5%), as compared to variant frequencies of approximately 60 to approximately 85% in the non pan-DENV sequence regions. We further showed that the majority of the conserved sequences were immunologically relevant: 34 contained numerous predicted human leukocyte antigen (HLA) supertype-restricted peptide sequences, and 26 contained T-cell determinants identified by studies with HLA-transgenic mice and/or reported to be immunogenic in humans. CONCLUSIONS/SIGNIFICANCE: Forty-four (44) pan-DENV sequences of at least 9 amino acids were highly conserved and identical in 80% or more of all recorded DENV sequences, and the majority were found to be immune-relevant by their correspondence to known or putative HLA-restricted T-cell determinants. The conservation of these sequences through the entire recorded DENV genetic history supports their possible value for diagnosis, prophylactic and/or therapeutic applications. The combination of bioinformatics and experimental approaches applied herein provides a framework for large-scale and systematic analysis of conserved and variable sequences of other pathogens, in particular, for rapidly mutating viruses, such as influenza A virus and HIV.

Single-tube nested PCR using immobilized internal primers for the identification of dengue virus serotypes.

Molecular techniques based on the detection of genomic sequences by reverse transcription (RT)-PCR, nested PCR, or real-time PCR have made possible the rapid diagnosis of dengue virus (DENV) infections, and these approaches have been accepted by clinical laboratories as the new standard method for the detection of dengue virus in acute-phase serum samples. One of these PCR-based assays, the two-step RT nested PCR (RT-NPCR) technique is used routinely in laboratories worldwide. In the present study, the two-step RT-NPCR as described by Lanciotti et al. [Lanciotti, R.S., Calisher, C.H., Gubler, D.J., Chang, G.J., Vorndam, A.V., 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J. Clin. Microbiol. 30, 545-551] was adapted to a novel single-tube nested PCR (STNPCR) format, which is less prone to cross-contamination and reduces reaction cost and time. When standards for each dengue serotype were tested, the detection limit of the STNPCR was at least 10 copies for DENV-1 and 100 copies for DENV-2 and DENV-3, whereas the detection limit for the two-step RT-NPCR was 100 copies for each serotype. Sera from 22 patients with confirmed DENV-3 infections and from 14 healthy individuals were then tested in the STNPCR format using the system described by Lanciotti et al. as the reference standard. The results indicated a sensitivity of 75.9% (CI 95%, 60.3-91.4) and a specificity of 100% for the RT-STNPCR. Although RT-STNPCR was less sensitive than the conventional two-step RT-NPCR for the detection of virus in serum samples, it was still adequately sensitive, and the advantages associated with a single-tube format may outweigh the somewhat lower assay sensitivity, making it useful for diagnosis in the field.

Fucosyltransferases in Schistosoma mansoni development.

Glycoconjugate-bound fucose, abundant in the parasite Schistosoma mansoni, has been found in the form of Fucalpha1,3GlcNAc, Fucalpha1,2Fuc, Fucalpha1,6GlcNAc, and perhaps Fucalpha1,4GlcNAc linkages. Here we quantify fucosyltransferase activities in three developmental stages of S. mansoni. Assays were performed using fluorophore-assisted carbohydrate electrophoresis with detection of radioactive fucose incorporation from GDP-[(14)C]-fucose into structurally defined acceptors. The total fucosyltransferase-specific activity in egg extracts was 50-fold higher than that in the other life stages tested (cercaria and adult worms). A fucosyltransferase was detected that transferred fucose to type-2 oligosaccharides (Galbeta1,4GlcNAc-R), both sialylated (with the sialic acid attached to the terminal Gal by alpha2,3 or 2,6 linkage) and nonsialylated. Another fucosyltransferase was identified that transferred fucose to lactose-based and type-2 fucosylated oligosaccharides, such as LNFIII (Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,3Galbeta1,4Glc). A low level of fucosyltransferase that transfers fucose to no-sialylated type-1 oligosaccharides (Galbeta1,3GlcNAc-R) was also detected. These studies revealed multifucosylated products of the reactions. In addition, the effects of fucose-type iminosugars inhibitors were tested on schistosome fucosyltransferases. A new fucose-type 1-N-iminosugar was four- to sixfold more potent as an inhibitor of schistosome fucosyltransferases in vitro than was deoxyfuconojirimycin. In vivo, this novel 1-iminosugar blocked the expression of a fucosylated epitope (mAb 128C3/3 antigen) that is associated with the pathogenesis of schistosomiasis.

Molecular characterization of a fucosyltransferase encoded by Schistosoma mansoni.

The glycans of schistosomes include many complex carbohydrates that contain fucose. Although the biological functions of these complex carbohydrates are not yet clearly understood, some of these structures are thought to play essential roles in the life cycle of the parasite. Here we present the molecular cloning and characterization of a fucosyltransferase of Schistosoma mansoni with a DNA sequence similarity of 84.6 and 63.7% to mouse and human fucosyltransferase type VII. Southern blot analysis of genomic DNA indicated that this S. mansoni fucosyltransferase is the product of a single gene. The schistosome cDNA sequence that we obtained contains an open reading frame encoding a protein of 351 amino acids with a predicted molecular size of 40.5 kDa. From the amino acid sequence, we predicted two potential N-linked and one O-linked glycosylation site. Western blot studies of extracts from stably transfected CHO cells showed a band corresponding to the schistosome fucosyltransferase at 50 kDa, suggesting that the enzyme is indeed glycosylated. We further demonstrated the expression and enzymatic activity of the fucosyltransferase in the transfected cells by immunofluorescence studies and flow microfluorimetric analysis, which indicated that the enzyme is capable of synthesizing the SLeX blood group determinant but not the LeX determinant in CHO cells. The identification of a fucosyltransferase type VII in schistosomes further underscores the importance of fucose-containing glycans in schistosome glycobiology.

Immobilization of xanthine oxidase on a polyaniline silicone support.

A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80% of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79% of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

Immobilization of xanthine oxidase on a polyaniline silicone support

A polyaniline silicone support to immobilize xanthine oxidase is proposed as a reactor coil to monitor the action of xanthine oxidase on hypoxanthine, xanthine and 6-mercaptopurine. A purified xanthine oxidase immobilized on this support lost 80 per cent of the initial activity after 12 min of use. Co-immobilization of superoxide dismutase and catalase increased the stability of immobilized xanthine oxidase so that the derivative maintained 79 per cent of its initial activity after 4.6 h of continuous use in which 1.5 mumol purine bases were converted by the immobilized enzyme system. There is no evidence of either polyaniline or protein leaching from the coil during 3 h of continuous use. When solutions (10 ml) of hypoxanthine, xanthine and 6-mercaptopurine were circulated individually through the xanthine oxidase-superoxide dismutase-catalase-polyaniline coil (1 mm internal diameter and 3 m in length, 3 ml internal volume) activities of 8.12, 11.17 and 1.09 nmol min-1 coil-1, respectively, were obtained. The advantages of the reactor configuration and the redox properties of the polymer, particularly with respect to immobilized oxidoreductases, make this methodology attractive for similar enzyme systems. This immobilized enzyme system using polyaniline-silicone as support converted 6-mercaptopurine to 6-thiouric acid with equal efficiency as resins based on polyacrylamide and polyamide 11.

Glucose biosensor using glucose oxidase immobilized in polyaniline.

A biosensor for glucose utilizing kinetics of glucose oxidase (EC 1.1.3.4.) was developed. The enzyme was immobilized on polyaniline by covalent bonding, using glutaraldehyde as a bifunctional agent. The system showed a linear response up to 2.2 mM of glucose with a response time of 2.5-4.0 min. In addition, the immobilized enzyme had a higher activity between pH 6.5 and 7.5. The system retained 50% of its activity after 30 d of daily use. The optical absorption spectra of the polyaniline/glucose oxidase electrode after glucose had been added to the buffer solution showed that the absorption band around 800 nm had changed considerably when glucose was allowed to react with the electrode. This optical variation makes polyaniline a very promising polymer for use as a support in optical sensor for clinical application.

Ascorbic acid biosensor using ascorbate oxidase immobilized on alkylamine glass beads.

A biosensor for ascorbic acid based on enzyme kinetics of ascorbate oxidase (E.C.1.10.3.3) was developed. The enzyme was extracted from Cucurbita maxima, or jerimun and immobilized by covalent bounding, using glutaradehyde as a bifunctional agent, on alkylamine glass beads, with and without enzyme active site protection. A low-cost, home-made oxygen electrode was applied as a transducer. The system has sensitivity from 62.5 up to 500 microM of ascorbic acid with satisfactory operation for more than 2 mo.